Identification of the replication-associated protein binding domain within the intergenic region of tomato leaf curl geminivirus.
The geminiviral replication-associated protein (Rep) is the only viral protein required for viral DNA replication. Tomato leaf curl virus (TLCV) Rep was expressed in Escherichia coli as a histidine-tagged fusion protein and purified to homogeneity in non-denaturing form. The fusion protein was used in in vitro binding experiments to identify the Rep-binding elements within the origin of replication of TLCV. Electrophoretic mobility shift assays demonstrated that the Rep binds specifically to a 120 bp fragment within the TLCV intergenic region. Fine resolution of the binding regions within the 120 bp fragment, using DNase I footprinting, demonstrated two footprints covering the sequences GCAATTGGTGTCTCTCAA and TGAATCGGTGTCTGGGG containing a direct repeat of the motif GGTGTCT (underlined). Our results suggest that the repeated motif is involved in virus-specific Rep-binding, but may not constitute the entire binding element. This is the first demonstration of geminivirus sequence elements involved in Rep-binding by direct protein-DNA interaction assays.
Akbar Behjatnia
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CSIRO, Plant Industry, Horticulture Research Unit, PO Box 350, Glen Osmond 5064, Adelaide, Australia.
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Ali Rezaian
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D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular; D018983:DNA Footprinting; D004265:DNA Helicases / Q000235:genetics* / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D004261:DNA Replication; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins; D017791:Geminiviridae / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D018551:Solanum lycopersicum / Q000821:virology; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012091:Repetitive Sequences, Nucleic Acid; D018741:Replication Origin; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism*; D014779:Virus Replication
D017931:DNA Primers; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D015534:Trans-Activators; D014764:Viral Proteins; C045264:replication initiator protein; D004265:DNA Helicases